Corneal cells expressing active agents and methods of use thereof

ABSTRACT

The invention relates to methods of modifying cells of corneal tissue to express an active agent, to modified corneal tissue, to vectors utilized in such methods and to methods of xeno- and allo-transplantation utilizing the modified corneal tissue. The method of modifying cells of corneal tissue to express an active agent involves exposing harvested corneal tissue to an effective concentration for transfection of an expression vector which comprises a nucleotide sequence encoding for the active agent for a period sufficient to allow infection, such that cells of said corneal tissue will express the active agent.

RELATED APPLICATION(S)

[0001] This application claims the benefit of Australian PatentApplication No. PRO695/00 filed on Oct. 11, 2000, the entire teachingsof which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] There are numerous diseases and disorders that can effect cornealtissue and which can, as a result, adversely effect or eliminate vision.For example, allergies, conjunctivitis, corneal infections, Fuchs'dystrophy (deterioration of corneal endothelial cells), varicella-zostervirus, iridocorneal endothelial syndrome, keratoconus, ocular herpes anda number of other conditions, as well as congenital cornealabnormalities can be responsible for corneal damage or irregularity thatmay affect vision. In an endeavour to restore sight or improve vision inpeople suffering from corneal abnormalities it has become particularlycommon to perform corneal transplant operations where the abnormalcorneal tissue is removed and replaced using fine sutures with normalcorneal tissue obtained from a donor. Although corneal transplantoperations enjoy a high rate of success there are nonetheless someproblems which can occur, such as rejection of the replacement corneaand ocular fibrosis or scarring. Even in the case of a successfulcorneal transplantation it is necessary for subsequent administration ofimmunomodulatory agents. Non-compliance by the patient with prescribeddosing regime of immunomodulating agents may give rise to tissuerejection. There is, accordingly, a need for improved means ofprolonging corneal graft survival and preventing tissue rejection aswell as for the provision of approaches for therapy of ocular infection,wounds and fibrosis and for therapy of other ocular disorders, forexample.

[0003] The cornea is a highly organised group of cells and proteinswhich unlike most tissue is clear, and does not contain blood vessels tonourish or protect against infection. The cornea receives nutritionalsupply from tears and the aqueous humor found in the anterior chamberlocated behind it. The cornea is composed of five basic layers, namelythe protective external epithelium, Bowman's layer which is locatedbelow the epithelial basement membrane and is composed of collagenfibers, the stroma which consists primarily of water and collagen and islocated beneath Bowman's layer; and Descemet's membrane located beneaththe stroma, which is composed of collagen fibers produced by theendothelial cells located in the lower endothelium. The endothelialcells are essential in maintaining clarity of the cornea by removingexcess fluid from the stroma.

[0004] Irreversible immunological rejection is the major cause of humancorneal graft failure (1), despite the immunologically privileged natureof the eye (2). The histological correlates of rejection include localupregulation of major histocompatability complex and adhesion molecules,an influx of mononuclear cells into the cornea and anterior chamber, andlocal production of some inflammatory cytokines (3-7). The major targetof corneal graft rejection is the corneal endothelium. Human (but notrodent) corneal endothelium is essentially amitotic (8), so that damageto the monolayer during graft rejection cannot be repaired.

[0005] Gene therapy has the potential to influence an allograft responsethrough local expression of a modulatory gene product withintransplanted donor tissue. The present inventors consider that thecornea may be uniquely amenable to such an approach because of its smallsize, which may allow modification of the whole tissue, and because ofthe ease with which a donor cornea may be manipulated in vitro andstored for a considerable period (for example, up to 28 days) prior totransplantation. The anatomical location and clarity of the cornea allowin vivo assessment of the entire graft in the post-operative period andloss of function is easy to detect. Furthermore, the cornea and anteriorchamber are at least partially immunologically privileged sites (2),which may allow the use of otherwise immunogenic or pro-inflammatoryvectors.

SUMMARY OF THE INVENTION

[0006] According to one embodiment of the present invention there isprovided a method of modifying cells of corneal tissue to express anactive agent comprising exposing harvested corneal tissue to aneffective concentration for infection of an expression vector whichcomprises a nucleotide sequence encoding for the active agent for aperiod sufficient to allow transfection, e.g., infection, such thatcells of said corneal tissue will express the active agent.

[0007] According to another embodiment of the present invention there isprovided a corneal tissue comprising cells modified to express an activeagent which is not expressed by normal corneal tissue or which followingmodification is expressed at elevated levels relative to normal cornealtissue.

[0008] In embodiments, the nucleotide sequence utilized in the methodsof the invention can be any type of nucleic acid, e.g., DNA or RNA. Thenucleic acid can be a full-length gene or an active portion of the gene.

[0009] In another embodiment of the invention there is provided acorneal tissue comprising cells modified to express an active agent,wherein modification is by exposing harvested corneal tissue to aneffective concentration for transfection, e.g., infection of anexpression vector which comprises a nucleotide sequence encoding for theactive agent, for a period sufficient to allow infection.

[0010] In a further embodiment of the invention there is provided amethod of improving corneal graft healing and/or prolonging graftsurvival comprising exposing harvested corneal tissue to an effectiveconcentration for infection of an expression vector which comprises anucleotide sequence encoding for an active agent for a period sufficientto allow transfection, e.g., infection, such that cells of said cornealtissue will express the active agent, and then transplanting the cornealtissue to an eye of a recipient.

[0011] In a still further embodiment of the invention there is providedan expression vector for use in modifying corneal tissue to express anactive agent not expressed by normal corneal tissue or which followingmodification is expressed at elevated levels relative to normal cornealtissue; the vector comprising a nucleotide sequence encoding for theactive agent.

[0012] Preferably the active agent is a peptide hormone, a cytokine oran analogue thereof. In a preferred embodiment of the invention thecytokine is an interleukin, an interferon or a growth factor, or ananalogue thereof. In preferred embodiments of the invention the cytokineis selected from the interleukins including IL-10, IL-4, the P-40component of IL-12 or from Bc12, interferon Gamma, interferon Alpha andTGF Beta.

[0013] In preferred embodiments of the invention the corneal tissue isharvested from a mammal, particularly preferably from a human.Preferably the recipient of transplanted corneal tissue is a mammal,particularly preferably a human. In a particularly preferred embodimentof the invention corneal tissue is harvested from a human andtransplanted to another human recipient.

[0014] In a preferred embodiment of the invention the corneal tissuecells modified are epithelial cells, stroma cells and/or endothelialcells. Particularly preferably, the modified cells are endothelialcells. In a preferred embodiment of the invention 5%, preferably atleast 10%, more preferably at least 20%, particularly preferably atleast 30% or at least 50% and most particularly preferably at least 70%of corneal endothelial cells in a sample of corneal endothelial cellsare modified by methods according to the invention.

[0015] In preferred embodiments of the invention the expression vectoris a viral, bacterial or plasmid vector. In particularly preferredembodiments of the invention the expression vector is anadeno-associated viral vector or an adenoviral vector.

[0016] Preferably the effective concentration for transfection, e.g.,infection is between about 1×10⁵ to 1×10¹⁰ particle forming units (pfu)per cornea. Particularly preferably the effective concentration forinfection is between about 5×10⁵ to 5×10⁸ pfu/cornea, more particularlypreferably between about 2×10⁶ and about 9×10⁷ pfu/cornea.

[0017] In a preferred embodiment of the invention the period sufficientto allow transfection, e.g., infection is between about 1 minute andabout 48 hours, particularly preferably between about 10 minutes and 24hours, more particularly preferably between about 30 minutes and 6 hoursand most particularly preferably between about 1 hour and about 3 hours.

[0018] In another preferred embodiment of the invention the expressionvector comprises DNA sequences encoding for two or more active agents.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings.

[0020]FIG. 1. Autoradiographs showing uptake of ³H thymidine inorgan-cultured ovine corneal endothelium, 3 days after deliberateinjury.

[0021]FIG. 1(A) shows uptake in endothelial cells (arrowed) at marginsof deliberate injury, magnification ×32;

[0022]FIG. 1(B) shows high power view showing mitotic figure (arrowed);magnification ×128.

[0023]FIG. 2. Effect of viral concentration and incubation time withvirus on transfection efficiency of the adenoviral vector Ad-lacZ forovine corneal endothelium, and stability of expression ofβ-galactosidase in endothelial cells of organ-cultured ovine corneas. Ineach instance, reporter gene expression was quantified by countingβ-galactosidase-positive cells in three representative areas per cornea.

[0024]FIG. 2(A) shows concentrations of Ad-lacZ from 6.6×10² to 6.6×10⁸pfu per cornea were used to transfect ovine corneas in vitro underotherwise identical conditions. Corneas were harvested 48 hours later.Each bar represents the mean percentage positive cells ±SD of countsfrom 3 to 6 corneas.

[0025]FIG. 2(B) shows ovine corneas were incubated with Ad-lacZ at6.6×10⁶ and 6.6×10⁷ pfu per cornea for 0.5-2.0 h. Corneas were harvested48 hours later. Each bar represents the mean percentage of positivecells ±SD from 3 organ-cultured corneas.

[0026]FIG. 2(C) shows duration of expression of β-galactosidase inorgan-cultured ovine corneas after transfection with 6.6×10⁶ Ad-lacz pfuper cornea for 2 hours. Corneas were harvested at the indicatedtime-points. Each point represents the mean percentage positive cells±SD of 3-14 corneas.

[0027]FIG. 3. Agarose 1.5% gel showing product for IL-10 and GAPDH insheep corneas transfected with Ad-IL-10 under optimal conditions andorgan-cultured for 21 days prior to RNA extraction and RT-PCR. Dilutionsof cDNA at 1/1, 1/10 and 1/100 dilutions were run in duplicate lanes.Lanes marked no DNA represent controls in which water replaced cDNA.

[0028]FIG. 4. Outcome of gene-modified penetrating corneal allografts insheep:

[0029]FIG. 4(A) shows rejecting unmodified allograft, day 29 post-graft;

[0030]FIG. 4(B) shows surviving IL-10-modified allograft, day 190post-graft.

DETAILED DESCRIPTION OF THE INVENTION

[0031] As indicated above the present inventors have devised an approachfor treating ocular disorders and demonstrated its efficacy in a sheepmodel. In the specifically exemplified embodiments of the presentinvention harvested corneal tissue has been modified to expressimmunomodulatory agents having the effect of prolonging corneal graftsurvival once implanted into a recipient, relative to the expectedsurvival time in the case where no immunomodulatory agent isadministered. However, the present invention has considerably broaderapplication than simply its use in association with corneal allo- orxeno-transplantation techniques. Methods according to the presentinvention may be adopted for treatment of other ocular disorders suchas, for example, the treatment of ocular wounds, infections or fibrosisor of other ocular diseases such as glaucoma, keratoconus, cornealdystrophies, corneal infections, tumours of the eye, proliferativelesions, pterygium and inflammatory disorders of the eye includingStevens-Johnson syndrome and mucous panphigoid. Reference to the term“treatment” is intended to include both therapeutic and prophylactictreatments.

[0032] One aspect of the invention relates to methods of modifying cellsof corneal tissue to express an active agent. As these active agents areto be expressed in the corneal tissue cells as a result of genetransfer, the active agents will of course constitute peptides,polypeptides or proteins, the expression of which can be encoded for bynucleotide, particularly sequences. Collectively, such active agents,regardless of the peptide sequence, maybe referred to herein as“peptides”. The active agents according to the invention may for exampleconstitute naturally occurring or synthetic peptide hormones, cytokinesor analogues thereof. By use of the term “analogue” it is intended toembrace modified forms of naturally occurring or synthetic peptidehormones or cytokines having physiological activity which may forexample be modified relative to the molecule upon which they are basedby the addition, deletion or substitution of single or multiple aminoacids.

[0033] Examples of active agents that may be expressed by the methodsand vectors according to the present invention include peptide hormonesand cytokines and analogues there of which may for example haveimmunomodulatory, anti infective, tissue regeneration, wound healing orfibrosis reduction activity. Cytokines that may be adopted in thepresent invention include those selected from the interleukins, theinterferons and the growth factors as well as analogues thereof.Specific examples of cytokines that may be adopted include IL-10, IL-4,the P-40 component of IL-12, Bcl2, interferon gamma, interferon alphaand TGF beta. It is to be understood however that these specificcytokines are mentioned as active agents by way of example only, andthat other peptide agents with useful activity may equally be adopted.

[0034] The corneal tissue to be modified according to the presentinvention will generally have been harvested from a donor, usually adonor mammal. Preferably the donor will be selected from the samespecies as the corneal transplant recipient and generally from a donorhaving matching tissue and/or blood types as the intended recipient, aswell understood in the art. There may, however, be circumstances, suchas if there is insufficient donor organ supply from members of the samespecies (allo-transplantation), where corneal tissue is harvested from adonor member of another species (xeno-transplantation). In the case ofxeno-transplantation the tissue donor may be an animal that has beengenetically modified to remove or reduce the impact of species specificimmunogenic differences. It may also be possible in future for cornealtissue to be produced by organ culture techniques which can thensimilarly be harvested for modification by methods according to thepresent invention.

[0035] Mammals from which corneal tissue maybe harvested and/or to whichcorneal tissue may be transplanted include, but are not limited to,humans, farm animals including cattle, sheep, goats, pigs, horses, etc.;captive wild animals including lions, tigers, deer, chimpanzees, apes,gorillas, baboons, etc.; domestic animals such as cats and dogs, etc, orlaboratory animals such as rabbits, mice, guinea pigs, rats and thelike. Preferably the corneal tissue is harvested from a human donor fortransplant to a human recipient. In the case of human corneal tissuedonors, the donor will generally be a person registered as an organdonor who has met an untimely death, and whose corneal tissue is in goodcondition. In the case of animal donors, the animal may be sacrificed inorder to harvest the corneal tissue or may in fact be sacrificed forother purposes such that the corneal tissue becomes available.

[0036] The corneal tissue will preferably be obtained from the donorwithin a relatively short period post mortem, preferably within three tofour hours and particularly preferably within the first hour. Theconditions under which the corneal tissue should be removed from thedonor and maintained prior to modification are a matter of routine andare well understood by persons skilled in the art. Naturally, the use ofa suitable tissue culture media is required to maintain the tissue in ahealthy state prior to modification and transplantation. Preferablymodification of the corneal tissue will be conducted within a matter ofa few hours from harvesting of the tissue, although it is possible tomaintain corneal tissue under tissue culture conditions for up to about28 days.

[0037] The expression vector according to the present invention mayconstitute any of a wide variety of already known or even as yetunidentified types of expression vector, such as viral, bacterial orplasmid expressing vector systems. Examples of suitable viral vectorsinclude HSV, lentivirus, retroviral vectors and adeno-associated viralvectors. Preferred vectors are adenoviral vectors. Naturally, theexpression vector adopted must be one which can transfect, e.g., infect,and result in protein expression in corneal tissue cells andparticularly cells of the corneal epithelium, stroma and/or endothelium.Preferably, expression of the active agent, through infection by theexpression vector, is within the endothelial cells and particularlypreferably the level of infection of these cells with the selectedexpression vector is such that active ingredient expression isdemonstrated in at least 5%, preferably at least 10%, more preferably atleast 20%, particularly preferably at least 30% or at least 50% and mostparticularly preferably at least 70% in a sample of corneal endothelialcells. The expression vector selected will of course include all of thefeatures required for expression of protein in a mammalian cell. Forexample, preferred expression vectors will contain a molecular chimeracontaining the coding sequence of active agent or agents selected, anappropriate polyadenylation signal for a mammalian gene (i.e. apolyadenylation signal which will function in a mammalian gene), andsuitable enhancers and promoter sequences in the correct orientation.

[0038] In mammalian cells, normally two DNA sequences are required forthe complete and efficient transcriptional regulation of genes thatencode messenger RNAs in mammalian cells: promoters and enhancers.Promoters are located immediately upstream (5′ from the start side oftranscription. Promoter sequences are required for accurate andefficient initiation of transcription. Different gene-specific promotersreveal a common pattern or organisation. A typical promoter includes anAT-rich region called a TATA box (which is located approximately 30 basepairs 5′ to the transcription initiation start site) and one or moreupstream promoter elements (UPE). The UPEs are a principle target forthe interaction with sequence-specific nuclear transcription factors.The activity of promoter sequences is modulated by other sequencescalled enhancers. The enhancer sequence may be a great distance from thepromoter in either upstream (5′) or downstream (3′) position. Hence,enhancers operate in an orientation- and position-independent manner.However, based on similar structural organisation and function that maybe interchanged the absolute distinction between promoters and enhancersis somewhat arbitrary. Enhancers increase the rate of transcription fromthe promoter sequence. The necessary machinery required for cellularexpression of the active agent or agents must of course be located inthe appropriate orientation with regard to the nucleotide sequence(preferably DNA) that encodes for the active agent or agents that hasbeen inserted into the expression vector by the use of routine molecularbiology techniques, such as, for example, as further explained inAusubel et al. (1987) in: Current Protocols in Molecular Biology, WyleInterscience (ISBN 047150338) the disclosure of which is incorporated byreference herein in its entirety. Also mentioned by way of reference inrelation to preparation of expression vectors, the disclosure of whichis included herein by reference is He et al., “A simplified system forgenerating recombinant adenovirus”, Proc. Nat. Acad. Sci. (1998) 95:2509-2514. The expression vector can appropriately include a suitablenuclear localisation signal and will be propagated in any permissivecell line. Permissive cell lines, mentioned by way of example only,include E1A and E1B trans-complementing 293 cells. Other cell lines willequally be useful for propagation of expression vectors according to theinvention, as would clearly be understood by persons skilled in the art.

[0039] The exposure of corneal tissue to expression vectors according tothe invention will be in a manner that will allow infection by theexpression vector of the corneal tissue cells. The exposure of thecorneal tissue to the expression vector can simply be by including theexpression vector into the corneal tissue culture media. Other means ofexposure such as via direct injection of the expression vectors into thecorneal tissue or via high velocity bombardment may also be adopted,although care should be taken to avoid damage to the corneal tissue. Toensure adequate levels of infection of corneal cells with the expressionvector it is necessary for an effective concentration for infection ofthe expression vector to be utilised. For example, concentrations ofbetween about 1×10⁵ and about 1×10¹⁰ particle forming units (pfu) percornea may be adopted. Preferably the effective concentration is betweenabout 5×10⁵ and 5×10⁸ pfu/cornea, particularly preferably between about2×10⁶ and about 9×10⁷ pfu per cornea. It is also important that theexposure of the corneal tissue to the expression vector is for a periodsufficient to allow infection, such as for example between about 1minute and about 48 hours, preferably between about 10 minutes and 24hours, more preferably between about 30 minutes and about 6 hours andmost preferably between about 1 hour and about 3 hours. This can, forexample, be achieved by simply introducing the expression vector intothe corneal tissue culture media and then changing the media to removeany remaining non-infected vector after the appropriate period,optionally with one or more washing stages.

[0040] The active agent which the corneal cells are modified to expresscan be one which is not expressed by normal corneal tissue or which,following modification is expressed at elevated levels relative to thoseof normal corneal tissue.

[0041] Also encompassed within the scope of the present invention areprocesses of improving corneal graft healing and/or prolonging graftsurvival involving the use of corneal tissue modified according tomethods discussed above. When referring to “improving corneal grafthealing” and “prolonging graft survival” these terms are intended to berelative to the rate and extent of healing and the duration of graftsurvival expected by conducting corneal allo-transplantation, withoutthe administration to the patient of other graft healing orimmunomodulatory agents.

[0042] Also included within the scope of the invention are theexpression vectors prepared for use in methods according to theinvention which allow for expression of active agents within cornealtissue cells, as well as the corneal tissue which comprises cellsmodified to express active agents.

[0043] The entire teachings of all patents, patent applications, booksand references described or cited herein are hereby incorporated byreference in their entireties.

[0044] The present invention will now be described further, by way ofexample only, with reference to the following examples:

EXAMPLES

[0045] The inventors selected a model of orthotopic cornealtransplantation in the outbred sheep, a relevant preclinical model inwhich unmodified corneal allografts undergo rejection at three weekspost-operatively in a manner that is very similar at a clinical level tohuman corneal graft rejection (9). Adenoviral vectors have already beenshown to be capable of transferring reporter genes into cornealendothelium of various species (10-13) and the use of liposomal agentshas also previously been explored (14, 15). Given that the mitoticpotential of sheep corneal endothelium was unknown, the replicativecapacity of this tissue was first examined, to allow an informed choiceof the vector for gene therapy to be made. The immunomodulatory cytokineIL-10, which down-regulates cell-mediated immune responses under somecircumstances (16, 17), was chosen as the candidate gene product forregulation of allograft rejection by ex vivo gene therapy.

[0046] Materials and Methods

[0047] Ovine Corneal Organ-culture.

[0048] Fresh sheep eyes obtained within 3 hr of donor death from a localabattoir (Lobethal Abattoirs, Lobethal, SA, Australia) weredecontaminated for 3 min in 10% w/v povidone-iodine (FauldingPharmaceuticals, Salisbury, SA, Australia) and underwent two washes byimmersion in sterile 0.9% w/v NaCl. A limbal incision was made with ascalpel blade and the cornea with a 2 mm scleral rim was removed withcorneal scissors. Corneas were organ-cultured in 15 ml complete medium(HBEPES-buffered RPMI medium (ICN, Costa Mesa, Calif., USA) supplementedwith 10% v/v heat-inactivated (56° C., 30 min) fetal calf serum (FCS),100 IU/ml penicillin, 100 μg/ml streptomycin, 2.5 μg/ml amphotericin Band 2 mM L-glutamine (all from Gibco BRL, Gaithersburg, Md., USA) at 32°C. in air for up to 28 days. Medium was changed twice weekly.

[0049] Evaluation of the Mitotic Potential of Ovine Corneal Endothelium.

[0050] A 4 mm long central cross-shaped defect was produced with a 27gauge needle on endothelial monolayers of fresh ovine corneas. Corneaswere then placed in sterile shallow wells, endothelium facing upward.500 μl complete medium containing 25 μCi 6-³H thymidine (TRA61;Amersham, Little Chalfont, Buckinghamshire, UK) was placed in thecorneal cup for 5 hr at 32° C. The solution was then diluted to a totalvolume of 3 ml with complete medium containing no isotope and to 10 mltotal after 24 hr. After 3 days, corneas were harvested, fixed in 3:1absolute ethanol:glacial acetic acid at room temperature for 24 hr andtransferred to 70% ethanol for a further 24 hr. Corneal endothelium wasremoved by blunt dissection through the stroma, mounted ongelatin-coated slides and air-dried for 2 hr. Flat-mounts were coatedwith LM-1 photographic autoradiography emulsion (Amersham, LittleChalfont, Buckinghamshire, UK), exposed at 4° C. for 4 weeks andprocessed according to the manufacturer's protocol. The flat-mounts werestained with Giemsa and mounted in Depex (BDH Chemicals, Kilsyth, VIC,Australia). As negative controls, corneas were injured and incubated in³H thymidine-free medium, and uninjured corneas were incubated with andwithout ³H thymidine. Corneal epithelial flat-mounts prepared fromcorneas incubated as above with the epithelial surface in contact withtritiated thymidine-containing medium were used as a positive control.

[0051] Transfection of Ovine Corneal Endothelium with AdenoviralVectors.

[0052] The replication deficient E1-, E3-deleted adenovirus type 5vectors encoding E. coli lacZ under the transcriptional control of theCMV promoter (Ad-lacZ), or containing an empty plasmid (Ad-mock), orencoding full-length ovine IL-10 (Ad-IL-10) or P-40 subunit of IL-12(Ad-P40-IL-12) (cDNA sequence provided by Dr S. Swinburn, HaematologyDepartment, Flinders Medical Centre, South Australia) where preparedfollowing the approach as described in Hu et al. as referenced above.cDNA sequences for these species are available on public databases. TheAd-lacZ construct contained a nuclear localization signal. Vectors werepropagated in E1A, E1B trans-complementing 293 cells following standardprotocols (18-20). In order to determine optimal viral concentration forinfection of corneal endothelial cells, corneas were infected withconcentrations of Ad-lacZ ranging from 6.6×10²-6.6×10⁸ plaque formingunits (pfu) per cornea in complete medium. Control corneas wereuninfected or similarly infected with Ad-mock. Optimal infection timewas determined by incubation of the corneas with 6.6×10⁶ and 6.6×10⁷ pfuAd-lacZ per cornea for 0.5, 1, 1.5 and 2 hours; the vector was thendiluted out and the corneas were re-incubated for a further 48 hr in 15ml complete medium. To examine duration of reporter gene expression,corneas infected with 6.6×10⁷ pfu per cornea for 2 hours at roomtemperature were organ-cultured for 2 days (n=14), 3 days (n=6), 6 days(n=6), 7 days (n==1), 10 days (n=5), 13 days (n=5), 14 days (n=1), 16days (n=3), 21 days (n=4), and 28 days (n=3). After incubation, allcorneas were processed for lacZ reporter gene expression.

[0053] Detection of lacZ Reporter Gene Expression.

[0054] Prior to processing, corneas were fixed in 2.5% formaldehyde and0.25% glutaraldehyde in Dulbecco's A phosphate-buffered saline (PBS) for15 min on ice followed by two 15 min washes in PBS on ice to inactivatethe viral vector and inhibit endogenous -galactosidase (21). Expressionof E. coli β-galactosidase was detected using 2.5 ml/cornea of asolution of 1 mg/ml 5-bromo-4-chloro-3-indoxyl-β-D-galactoside (ICN,Costa Mesa, Calif., USA), N-dimethylformamide (BDH Chemicals, Kilsyth,VIC, Australia), 2 mM MgCl₂, 5 mM K₄Fe(CN)₆, 5 mM K₃Fe(CN)₆ in PBS-2 (16mM Na₂HPO₄, 4 mM NaH₂PO₄.2H₂O, 120 mM NaCl), pH 7.0 at 32° C. for 18 hrin the dark. After a 10 min wash with 20 ml water per cornea, a modifiedsilver stain to stain endothelial cell boundaries was performed byapplication of 1% w/v AgNO₃ for 1 min and subsequent exposure to light(22). The endothelium was surgically removed using a 23 gauge needle andtoothed forceps, and mounted in Kaiser's glycerol jelly (12.5% w/vgelatin, 87.5% v/v glycerin) on chrome-alum subbed slides. To detect E.coli β-galactosidase in 293 cells, cells were washed twice with PBS,fixed on ice with 0.25% glutaraldehyde in PBS for 5 min and washed twicewith ice-cold PBS. Staining was then performed as described above.

[0055] Quantification of lacZ Expression.

[0056] To quantify the number of cells expressing the reporter gene,corneal endothelial flat-mounts were examined by light microscopy andphotographed on 35 mm slide film at standard magnifications. The slideswere projected at a standard distance and magnification. Total numbersof endothelial cells and lacZ positive cells were counted within framesof known dimension. For each cornea, three areas on each of twodifferent slides taken of representative areas of the flat-mount werecounted, and the mean and standard deviation (SD) calculated.

[0057] Detection of IL-10 mRNA in Transfected Ovine Corneas.

[0058] Fresh corneas prepared as described above were infected with4.5×10⁶ pfu Ad-mock or Ad-IL-10 for 2 hr or were incubated in mediumwithout viral vector. They were then incubated in 3 ml complete mediumat 32° C. in air for 24 hr, after which a further 2 ml of completemedium was added and organ-culture was continued. At various time pointsthereafter, a central 8 mm diameter full-thickness disc of cornea wastrephined and snap-frozen in liquid nitrogen. Each disc was pulverisedin a pre-chilled stainless steel mortar and pestle. Total RNA wasextracted with Total RNA Extraction Reagent (Advanced BiotechnologiesLtd., Surrey, UK), treated with DNAse (GlassMax MicroIsolation Kit, LifeTechnologies, Melbourne, VIC, Australia) and reverse-transcribed using acommercially-available first-strand cDNA synthesis kit (AmershamPharmacia Biotech UK Limited, Buckinghamshire, England) according to themanufacturers' recommendations. To control for residual ovine genomic orviral DNA contamination, samples were subjected to the samereverse-transcription step after inactivation of the reversetranscriptase at 95° C. for 60 min. Dilutions of cDNA were amplified in25 μl total volume by PCR. The reaction mixture for L-10 and β-actin was10 mM Tris-HCL (pH 8.3), 0.15M KCl (Perkin Elmer Roche MolecularSystems, Branchburg, N.J., USA), 0.2 mM of each dNTP (Amersham PharmaciaBiotech UK Limited, Buckinghamshire, England), 1.5 mM MgCl₂, 1 mM ofeach primer, 1 unit AmpliTaq-Gold (all from Perkin Elmer Roche MolecularSystems, Branchburg, N.J., USA) and 5 μl of sample. The reaction bufferfor glyceraldehyde 3-phosphate dehydrogenase (GAPDH) contained 2 mMMgCl₂ but was otherwise identical. Primer sequences amplified a 307 basepair region for IL-10 (5′-GCAGCTGTACCCACTTCCCA-3′,5′-AGAAAACGATGACAGCG-3′), a 317 base pair region for -actin(5′-ATCATGTTTGAGACCTTCAA-3′, 5′-CATCTCTTGCTCGAAGTCCA-3′), and a 527 basepair region for GAPDH (5′-ACCACCATGGAGAAGGCTGG-3′,5′-CTCAGTGTAGCCCAGGATGC-3′). After one cycle of 15 min at 94° C., 40cycles of amplification were performed, each consisting of annealing at55° C. for 30 sec, extension at 72° C. for 30 sec and 94° C. for 1 min,final extension at 55° C. for 30 sec, 72° C. for 20 sec and 35° C. for10 sec. Amplified products were electrophoresed on 1.5% agarose w/vgels.

[0059] Orthotopic Corneal Transplantation in Sheep.

[0060] Adult female Merino-cross breed sheep were acclimatised in groupsof at least two animals for at least one week in indoor pens and werefed water ad libitum and chaff supplemented with lucerne hay. Twelve mmdiameter penetrating corneal transplantation was performed as previouslydescribed (9) in the right eye only. Post-operative care and inspectionwere as previously described and every graft was examined at theslit-lamp each day. Groups of sheep received unmodified corneal grafts,corneas infected with Ad-mock, or corneas that had been infected withAd-IL-10 or Ad-P40-IL-12 according to optimised procedures. The order inwhich sheep were grafted was random amongst all groups. Rejection wasdefined as reported previously (9). In several sheep with long-survivingcorneal grafts, attempts were made to induce rejection by placement of8-0 braided silk sutures into the graft under general anaesthetic, as aninflammatory stimulus. Approval for all experimentation was obtainedfrom the institutional Animal Welfare Committee.

[0061] End-point Histology of Corneal Allografts.

[0062] Corneal tissue was fixed in buffered formalin, embedded inparaffin wax, cut at 8 μm and stained with haematoxylin and eosin.

[0063] Immunoperoxidase Staining of Corneal Allografts.

[0064] Hybridoma culture supernatants containing mouse mAbs to sheepcell-surface determinants were obtained from the Department ofVeterinary Science, University of Melbourne, Parkville, VIC, Australiaand included: SBU 41.19, anti-MHC class I monomorphic epitope (23); SBU28.1, anti-MHC class II monomorphic epitope (24); SBU 1-11-32,anti-CD45/leucocyte-common (unrestricted) antigen (25); SBU 44.38,anti-CD4 and SBU 38.65, anti-CD8 (26, 27); SBU 20.27, anti-CD1 (28); andSBU 72.87, anti-CD11a/LFA-1 (29). Culture supernatants from thehybridomas P3×63Ag8 (IgG1 isotype; European Collection of Animal CellCultures, Porton Down, Wiltshire, UK) and SAL5 (IgG2a isotype; gift ofDr L Ashman, IMVS, Adelaide, SA, Australia) were used as negativecontrols. Grafted eyes were harvested immediately post-mortem and thecornea excised, fixed, stained and scored as previously described (9).

[0065] Adenoviral Antibody Titres Influids from Sheep with CornealAllografts.

[0066] Immediately post-mortem, anterior chamber fluid was collected andsnap-frozen at −80° C. Venous peripheral blood was also collected, theserum separated and similarly snap-frozen. Antibody titres to adenoviruswere determined by a standard complement fixation test in the localreference laboratory using reagents from Biowhittaker NorthfieldLaboratories, Adelaide, SA, Australia.

[0067] Statistical Analysis of Data.

[0068] Corneal graft survival data were analysed with the Mann-WhitneyU-test, corrected for ties.

[0069] Results

[0070] Replicative Capacity of Ovine Corneal Endothelium.

[0071] Ovine corneal endothelium was deliberately injured and thecorneas organ-cultured in the presence of ³H thymidine. The site ofinjury was still clearly visible at the light microscope on cornealendothelial flat-mounts harvested after 3 days in organ-culture. Uptakeof ³H thymidine into the nuclei of the occasional endothelial cell closeto the site of the injury was observed (FIG. 1A) and very rare mitoticFigures were identified (FIG. 1B). Uptake of ³H thyrnidine was limitedto the close vicinity of the injury: no uptake occurred in the cornealperiphery. Uninjured corneas (negative control) showed no uptake of ³Hthymidine and corneas incubated with the epithelial surface in contactwith isotope-containing solution (positive control) showed substantialuptake (not shown). The data suggested that the replicative capacity ofovine corneal endothelium was very limited and that, for example,replication-deficient adenoviral virus (which remains episomal) wouldthus be a suitable vector for gene transfer to ovine endothelium.

[0072] Adenoviral-mediated Reporter Gene Transfer to Ovine CornealEndothelium.

[0073] Ovine corneas were transfected in vitro with Ad-lacZ. At6.6×10²-6.6×10⁴ pfu/cornea, single β-galactosidase-positive cells wereobserved scattered over the endothelial monolayer. Increasing the virusconcentration increased the number of β-galactosidase-positive cells toa maximum of approximately 50% (FIG. 2A), although a drop in expressionwas observed at 6.6×10⁸ pfu/cornea. A concentration of 6.6×10⁶⁻⁷pfu/cornea was judged to yield optimal expression. None of the negativecontrols (no virus applied, Ad-mock applied) showed expression of-galactosidase at any time. Reporter gene expression was observed onlyin corneal endothelium, not in stromal keratocytes. No visible toxiceffects on the cornea were observed at any virus concentration. Theinfluence of varying the time that the vector was in contact withcorneal endothelium was investigated at 6.6×10⁶ and 6.6×10⁷ pfu/cornea(FIG. 2B): about 30% of cells were infected within the first hour, thenumber of positive cells increasing to about 50% at 2 hours. Duration ofreporter gene expression was examined in a time-course experiment using6.6×10⁷ pfu/cornea and an infection time of 1.5 hr: 30% cells expressedβ-galactosidase after 24 hr, rising to approximately 70% at day 6, andexpression remained at this level for the 28-day observation period(FIG. 2C).

[0074] Detection of IL-10 mRNA in IL-10 Gene-modified Organ-culturedOvine Corneas.

[0075] Ad-IL-10 was used to transfer the gene encoding ovine IL-10 intosheep corneal endothelium using conditions optimised for reporter geneexpression, and the corneas were cultured in vitro for up to 21 days.Reverse transcription PCR was used to detect presence of mRNA for IL-10;β-actin and GAPDH served as housekeeping controls. No amplification ofgenomic or adenoviral IL-10 was observed in controls in which thereverse transcriptase had been inactivated after DNAse-I treatment ofthe isolated RNA preparations. Specific mRNA for ovine IL-10 wasobserved 24 hr after adenoviral infection and at various time pointsthereafter (Table 1), and could still be detected after corneas had beenorgan-cultured for 21 days (FIG. 3).

[0076] Orthotopic Transplantation of Gene-modified Donor Corneas inOutbred Sheep.

[0077] The Ad-IL-10 and Ad-P40-IL-12 vectors were used to infect cornealendothelium of donor corneas immediately prior to orthotopic cornealtransplantation in outbred sheep (FIG. 4). Controls included unmodifieddonor corneas and corneas infected with Ad-mock. Corneal graft survivaldata are shown in Table 2: corneal grafts modified by insertion of thegene encoding IL-10 into the donor endothelium survived significantlylonger than unmodified controls (p =0.019) or the combined unmodifiedand mock virus-infected control groups (p=0.011). There was nodifference in the time at which host vessels crossed thegraft-host-junction amongst the groups (p>0.05). Longer survival wasalso demonstrated in corneas modified by c-section of the genes encodingthe P-40 subunit of L-12 (median 45 days). Post-operative inflammationwas no more severe, and lasted for no longer, in the groups receivingadenovirus-treated corneas compared with the controls. End-pointhistology in sheep that showed clinical rejection of their grafts showeda similar picture in all instances: there was no difference amongst theexperimental groups. Similarly, immunoperoxidase staining showed thatrejecting gene-modified corneas contained a cellular infiltrate similarto that seen in rejecting unmodified or mock virus-infected grafts, witha substantial infiltrate of both CD4-positive and CD8-positive cells. Noantibody to adenovirus was detectable in anterior chamber fluid or serumfrom 2 sheep at post-mortem.

[0078] Attempts were made to induce rejection in two sheep from theIL-10 group with gene-modified, long-surviving (>150 days) cornealgrafts by placement of silk sutures into the graft at 196 and 303 dayspost-graft, respectively. In both cases, inflammation of the graftensued and rejection occurred within two weeks, indicating that neitherrecipient was tolerant of the graft. Immunohistochemistry indicated thatthe infiltrate in these rejected grafts was similar to that seen inunmodified grafts.

[0079] Discussion

[0080]³H thymidine is incorporated into DNA by proliferating cells andcan be visualised by autoradiography. This method has been usedpreviously (30-33) to investigate the mitotic activity of cornealendothelium in many species. In the sheep cornea, uninjured endotheliaincubated with ³H thymidine did not take up the isotope. Localizeduptake into single endothelial cells was observed after deliberateinjury, but proliferation was insufficient to cover the defect overthree days. Our data suggest that ovine corneal endothelium has a verylimited mitotic potential: some proliferation can be induced by atriggering event, but does not otherwise occur. For practical purposes,then, ovine corneal endothelium may be considered essentially amitotic.This resembles the situation in human and cat, where little mitosisoccurs and defects are mainly covered by gradual sliding and enlargementof existing cells (30-33).

[0081] Replication-deficient adenoviruses, which remain episomal and donot integrate into the host genome, are suitable vectors for genetherapy of amitotic cells. A replication-defective adenovirus proved anefficient vector for gene transfer to 70-80% of ovine cornealendothelial cells. Optimal expression of the reporter gene in vitro wasobtained with 6.6×10⁶ pfu per cornea. Given that the sheep corneacontains approximately 8×10⁵ endothelial cells, 6.6×10⁶ pfu represents amultiplicity of infection of >10 virions per cell. Infection at higherconcentrations of the vector was less efficient, but no obvious toxiceffects were apparent at any viral concentration. The optimalconcentration was similar to that found by other authors for infectionof rabbit corneal endothelium with adenoviral vectors (13-15). Otherauthors have observed reporter gene expression in 7% of rabbit cornealendothelial cells using Lipofectamine (14), and in a relatively smallproportion of bovine endothelial cells using dioleoylphosphatidylethylanolamine (34). More recently, George and hiscolleagues have demonstrated that activated polyamidoamine dendrimers, anovel class of non-viral agent, can successfully be used to transfer agene into 6-10% of rabbit and human corneal endothelial cells (35).Adenovirus, however, appears significantly more efficient in achievinggene transfer than are non-viral agents.

[0082] Adenovirus binds to surface receptors and enters the cell byendocytosis via clathrin-coated vesicles, a fast process (36, 37). DNAreplication in replicative adenoviruses starts approximately 8 hoursafter infection (38). In the sheep cornea, most adenoviral infectionoccurred within the first hour, but a delay of 5-6 days was observedbefore reporter gene expression was maximal. A time lag of 3-7 days forlacZ expression driven by either the CMV or RSV promoter has beenobserved after adenoviral transfer to human corneal endothelium (12, 14,15). We observed lacZ expression in the sheep corneal endothelium to bestable for 4 weeks in vitro. Investigators working in the rabbit havefound expression of lacZ for 3-4 weeks in in vitro experiments (38), butfor only 1-2 weeks after orthotopic corneal transplantation (10). Theprotein β-galactosidase has a half-life of 2 weeks in neurons (39) butshorter expression has been observed in other tissues such asrespiratory epithelium (40), possibly due to gene silencing by promoterextinction (41). In the absence of a monoclonal antibody specific forovine IL-10, expression of IL-10 product in ovine corneal endothelialcells was assessed indirectly by detection of mRNA for IL-10 intransfected, organ-cultured corneas. We were able to detect IL-10 mRNAin ovine corneal endotheliun for at least 3 weeks in vitro.

[0083] In the absence of immunosuppression, corneal graft rejection inthe sheep occurs at approximately three weeks post-operatively (9). Wetherefore reasoned that expression of a transferred gene encoding animmunomodulatory cytokine for 3-4 weeks in vivo might be sufficient tomodulate graft rejection. Inflammatory responses in the eye followingreporter gene transfer have been reported previously (13), and similarfindings have been observed in other immunoprivileged tissues such asbrain (42). However, we considered it possible that expression of animmunomodulatory cytokine in a privileged site such as the eye might besufficient to ameliorate any local immune responses to the viral vector,as well as to the allograft. Interestingly, Qin and colleagues havepreviously reported modulation of the immune response to bothalloantigen and adenovirus antigens in a murine cardiac allograft modelfollowing adenoviral-mediated gene therapy with viral IL-10 (43).

[0084] Obvious toxicity that could be attributed to the use of theadenoviral vector was absent. The adenoviral construct used to deliverthe target gene to donor corneal endothelium did not elicit a measurableantibody response in the sheep after corneal transplantation, and didnot induce noticeable ocular inflammation over the time-course of theexperiment. Host vessels extended from the limbus towards all cornealgrafts at the same rate, irrespective of the experimental group. In mostanimals that received gene-modified donor corneas, neovascularizationwas not accompanied by corneal graft rejection and the corneal vesselsin these sheep did not maintain patency.

[0085] In our experiments, gene transfer of IL-10 to the donor corneaimmediately prior to transplantation prolonged corneal allograftsurvival to a significant extent in the cohort as a whole. In two cases,graft survival was prolonged indefinitely (>150 days). Allograftsurvival was also prolonged in the group where corneal tissue wasmodified with P-40 of L-12. It is notable that these result wereobtained without the use of any other immunosuppressive therapy at alland in particular, without use of topical glucocorticosteroid. However,some sheep with IL-10-modified donor corneas rejected their graftswithin the same time-frame as did the control animals. In these animals,graft rejection was indistinguishable at either a macroscopic ormicroscopic level from that observed in the controls. In particular, theextent and composition of the leucocytic infiltrate was similar in allcases and there was no obvious difference in expression of MHC class Ior II molecules within the graft. That tolerance was not induced in thelong-survivors is evinced by the observation that these animals didreject their grafts after deliberate application of an inflammatorystimulus to the graft. We hypothesize that expression of 10 by cornealendothelium was sufficient to modulate or significantly delay rejectionin the majority of animals, but that rejection overwhelmed theimmunomodulation in a minority of recipients.

[0086] Both cellular (44-46) and viral IL-10 (43, 47-49) have beenreported to prolong allograft survival and to modulate chronic rejectionin a variety of small animal models, and IL-10 gene knock-out mice showdecreased cardiac allograft survival and increased evidence of chronicrejection (50, 51). However, in at least one report, systemicadministration of murine IL-10 was shown to exacerbate murine cardiacallograft rejection (52), and further, subconjunctival and systemicadministration of various doses of murine IL-10 has been shown to beineffective in prolonging corneal graft survival in the rat (53). Theeffect of IL-10 on allograft survival appears to be dependent upon bothtiming of administration (45) and upon dose (46). Mouse and human IL-10may not be entirely homologous with respect to function: in particular,the former can be immunostimulatory for murine T cells (16), a propertynot shared by human IL-10 or viral IL-10, although it has been suggestedinformally that endotoxin levels in various cytokine preparations mayhave affected some results. Ovine IL-10 has been shown to inhibitinflammatory cytokine production by sheep macrophages (54) and webelieve it may have functional properties akin to human IL-10.

[0087] In summary, we report that delivery of genes encoding animmunomodulatory cytokines, mammalian IL-10 and P-40 subunit of IL-12,into donor corneal endothelium prior to transplantation results insignificant prolongation of corneal allograft survival in an outbredmodel in which the endothelium is essentially non-replicative, and inwhich rejection appears very similar to human corneal graft rejection atboth clinical and histological levels.

[0088] The present invention has been described by way of example onlyand it should be recognised that modifications and/or alterations to thespecific aspects of the invention which would be apparent to personsskilled in the art based on the disclosure herein, are also consideredto fall within the spirit and scope of the invention. TABLE 1 Detectionof mRNAs for ovine IL-10 or housekeeping genes β-actin and GAPDH inovine corneas after infection with adenoviral vectors^(a) Corneasinfected with:^(a) Time after Primer medium infection identity controlAd-mock^(b) Ad-IL-10^(c)  2 hours IL-10 −^(d) − (+)^(d) β-actin/GAPDH+^(d) + +  3 days IL-10 − − + β-actin/GAPDH + + +  7 days IL-10 − − +β-actin/GAPDH + + + 10 days IL-10 NT^(e) − + β-actin/GAPDH NT + + 14days IL-10 NT − + β-actin/GAPDH NT + + 21 days IL-10 NT − +β-actin/GAPDH NT + +

[0089] TABLE 2 Survival of control and gene-modified orthotopic cornealgrafts in outbred sheep transplanted with unmodified donor corneas, orwith corneas transfected before transplantation with Ad-mock, or withcorneas transfected before transplantation with Ad-IL-10, Ad-P40-IL-12or Ad-Il-4 Day vessels Donor cornea n crossed into graft^(a) Day ofrejection Unmodified 7 11, 10, 9, 8, 10, 9, 9 18, 19, 19, 20, 20, 22, 32median = 9 median = 20 Mock-transfected 3 5, 7, 8 19, 21, 29 median = 7median = 21 IL-10-transfected 9 5, 9, 9, 10, 10, 9, 19, 20, 30, 33, 55,66, 88, 11, 11, 9 >196, >300^(b) median = 9 median = 55 P-40 IL-12 9 —22, 23, 32, 36, 45, >50, transfected 93, 93, >100 median = 45

REFERENCES

[0090] 1. Williams K A, Muehlberg S M, Lewis R F, Coster D J on behalfof all contributors to the Australian Corneal Graft Registry (ACGR). Howsuccessful is corneal transplantation? A report from the AustralianCorneal Graft Register. Eye 1995; 9: 219.

[0091] 2. Griffith T S, Ferguson T A. The role of FasL-induced apoptosisin immune privilege. Immunology Today 1997; 18: 240.

[0092] 3. Williams K A, Coster D J. Clinical and experimental aspects ofcorneal transplantation. Transplantation Reviews 1993; 7: 44.

[0093] 4. Pepose J S, Gardner K M, Nestor M S, Foos R Y, Pettit T H.Detection of HLA class I and II antigens in rejected human cornealallografts. Ophthalmology 1985; 92: 1480.

[0094] 5. Holland E J, Chan C-C, Wetzig R P, Palestine A G, NussenblattR B. Clinical and immunohistologic studies of corneal rejection in therat penetrating keratoplasty model. Cornea 1991; 10: 374.

[0095] 6. Whitcup S M, Nussenblatt R B, Price F W, Chan C-C. Expressionof cell adhesion molecules in corneal graft rejection. Cornea 1993; 12:475.

[0096] 7. Sano Y, Osawa H, Sotozono C, Kinoshita S. Cytokine expressionduring orthotopic corneal allograft rejection in mice. hivest OphthalmolVis Sci 1998; 39: 1953.

[0097] 8. Tuft S J, Coster D J. The corneal endothelium. Eye 1990; 4:389.

[0098] 9. Williams KA, Standfield S D, Mills R A, et al. A new model oforthotopic penetrating corneal transplantation in the sheep: graftsurvival, phenotypes of infiltrating cells and local cytokineproduction. Aust N Z J Ophthalmol 1999; 27: 127.

[0099] 10. Larkin D F, Oral H B, Ring C J, Lemoine N R, George A J.Adenovirus-mediated gene delivery to the corneal endothelium.Transplantation 1996; 613: 363.

[0100] 11. Borras T, Tamm E R, Zigler J S Jr. Ocular adenovirus genetransfer varies in efficiency and inflammatory response. InvestOphthalmol Vis Sci 1996; 37: 1282.

[0101] 12. Budenz D L, Bennett J, Alonso L, Maguire A. In vivo genetransfer into murine corneal endothelial and trabecular meshwork cells.Invest Ophthalmol Vis Sci 1995; 36:2211.

[0102] 13. Oral H B, Larkin D F, Fehervari Z, et al. Ex vivoadenovirus-mediated gene transfer and immunomodulatory proteinproduction in human cornea. Gene Therapy 1997; 4: 639.

[0103] 14. Arancibia-Carcamo C V, Oral H B, Haskard D O, Larkin D F,George A J. Lipoadenofection-mediated gene delivery to the cornealendothelium: prospects for modulating graft rejection. Transplantation1998; 65: 62

[0104] 15. Fehervari Z, Rayner S A, Oral H B, George A J, Larkin D F.Gene transfer to ex-vivo stored corneas. Cornea 1997; 16: 459.

[0105] 16. De Waal Malefyt R, Yssel H, Roncarolo M-G, Spits H, de VriesJ E. Interleukin-10. Current Opin Immunol 1992; 4: 314.

[0106] 17. Moore K W, O Garra A, de Waal Malefyt R, Vieira P, Mosmann TR. Interleukin-10. Annu Rev. Immmunol 1993; 11:165.

[0107] 18. Graham F L, Prevec L. Manipulation of adenovirus vectors. In:Murray E J, ed Methods in Molecular Biology Gene Transfer and ExpressionProtocols, Vol. 7. Clifton, N.J.: The Humana Press, 1991: 109.

[0108] 19. Graham F, Smiley J, Russell W C, Nairn R. Characteristics ofa human cell line transformed by DNA from human adenovirus type 5. J GenVirol 1977; 36: 59.

[0109] 20. Kanegae Y, Makimura M, Saito I. A simple and efficient methodfor purification of infectious recombinant adenovirus. Japn J Med SciBiol 1994; 47: 157.

[0110] 21. Lojda Z, Gossrau R, Schiebler T H. In: EnzymhistochemischeMethoden. Berlin-Heidelberg: Springer Verlag, 1976: 128.

[0111] 22. Sherrard E. The corneal endothelium in vivo: its response tomild trauma. Exp Eye Res 1976; 22: 347.

[0112] 23. Gogolin-Ewens K J, Mackay C R, Mercer W R, Brandon M R. Sheeplymphocyte antigens (OLA). I Major histocompatibility complex class Imolecules. Immunology 1985: 56: 717.

[0113] 24. Puri N K, Mackay C R, Brandon M R. Sheep lymphocyte antigens(OLA). II Major histocompatibility complex class II molecules.Immunology 1985; 56: 725.

[0114] 25. Maddox J F, Mackay C R, Brandon M R. The sheep analogue ofleucocyte common antigen (LCA). Immunology 1985; 55: 347.

[0115] 26. Mackay C R, Maddox J F, Brandon M R. Surface antigens, SBU-14and SBU-T8, of sheep T lymphocyte subsets defined by monoclonalantibodies. Immunology 1985; 55: 739.

[0116] 27. Mackay C R, Maddox J F, Brandon M R. Three distinctsubpopulations of sheep T lymphocytes. Eur J hnmunol 1986: 16: 19.

[0117] 28. Mackay C R, Maddox J F, Gogolin-Ewens K J, Brandon M R.Characterization of two sheep lymphocytic differentiation antigens,SBU-T1 and SBU-T6. Immunology 1085; 55: 729.

[0118] 29. Grooby W L, Carter J K, Rao M M, et al. Use of an anti-LFA-1antibody allograft rejection in sheep. Transplant. Proc 1992; 24: 2304.

[0119] 30. Van Horn D L, Sendele D D, Seideman S, Buco P J. Regenerativecapacity of the corneal endothelium in rabbit and cat. Invest OphthamolVis Sci 1977; 16: 597.

[0120] 31. Simonsen A H, Sorensen K E, Sperling S. Thymidineincorporation by human endothelium during organ culture. Acta Ophthahnol1981; 59: 110.

[0121] 32. Tuft S J, Williams K A, Coster D J. Endothelial repair in therat cornea. Invest Ophthahnol Vis Sci 1986; 27: 1199.

[0122] 33. Treffers W F. 1982. Corneal endothelial wound healing.Nijmegen: Janssen Print, 1982.

[0123] 34. Pleyer U, Druegg A, Bertelmann E, et al. Liposome-mediatedgene transfer into corneal endothelial cells. Invest Ophthalmol Vis Sci1997: 38: S402.

[0124] 35. Hudde T, Rayner S A, Comer R M, et al. Activatedpolyamidoamine dendrimers, a non-viral vector for gene transfer to thecorneal endothelium. Gene Therapy 1999; 6: 939.

[0125] 36. Philipson L, Lonberg-Holm K, Pettersson U. Virus-receptorinteraction in an adenovirus system. J Virol 1968; 2: 1064.

[0126] 37. Seth P, Fitzgerald D, H, Willingham M, Pastan I. Evidencethat the penton base of adenovirus is involved in potentiation oftoxicity of Pseudomonas exotoxin conjugated to epidermal growth factor.Mol Cell Biol 1984; 4: 1528.

[0127] 38. Hoffman L M, Maguire A M, Bennett J. Cell-mediated immuneresponse and stability of intraocular transgene expression afteradenovirus-mediated delivery. Invest Ophthalmol Vis Sci 1997; 38: 2224.

[0128] 39. Bloom D C, Maidment N T, Tan A, Dissette V B, Feldman L T,Stevens J G. Long term expression of a reporter gene from latent herpessimplex virus in the rat hippocampus. Brain Res Mol Brain Research 1995;31: 48.

[0129] 40. Goldman M J, Litzky L A, Engelhardt J F, Wilson J M. Transferof the CFTR gene to the lung of nonhuman primates with E1-deleted,E2a-defective recombinant adenoviruses: a preclinical toxicology study.Hum Gene Therapy 1995; 6: 839.

[0130] 41. Palmer T D, Rosman G J, Osborne W R, Miller A D. Geneticallymodified skin fibroblasts persist long after transplantation butgradually inactivate introduced genes. Proc Natl Acad Sci USA 1991; 88:1330.

[0131] 42. Byrnes A P, Rusby J E, Wood M J, Charlton H M. Adenovirusgene transfer causes inflammation in the brain. Neuroscience 1995; 66:1015.

[0132] 43. Qin L, Ding Y, Pahud D R, Robson N D, Shaked A, Bromberg J S.Adenovirus-mediated gene transfer of viral interleukin-10 inhibits theimmune response to both alloantigen and adenoviral antigen. Hum GeneTher 1997; 8: 1365.

[0133] 44. Shinozaki K, Yahata H, Tanji H, Sakaguchi T, Ito H, Dohi K.Allograft transduction of IL-10 prolongs survival following orthotopicliver transplantation. Gene Therapy 1999; 6: 816.

[0134] 45. Boehler A, Chamberlain D, Xing Z, et al. Adenovirus-mediatedinterleukin-10 gene transfer inhibits post-transplant fibrous airwayobliteration in an animal model of bronchiolitis obliterans. Hum GeneTherapy 1998; 9: 541.

[0135] 46. Zou X M, Yagihashi A, Hirata K, et al. Downregulation ofcytokine-induced neutrophil chemoattractant and prolongation of ratliver allograft survival by interleukin-10. Surg Today 1998; 28:184.

[0136] 47. Qin L, Chavin K D, Ding Y, et al. Retrovirus-mediatedtransfer of viral IL-10 prolongs murine cardiac allograft survival. JImmunol 1996; 156: 2316.

[0137] 48. DeBruyne L A, Li K, Chan S Y, Bishop D K, Bromberg J S.Lipid-mediated gene transfer of viral IL-10 prolongs vascularizedcardiac allograft survival by inhibiting donor-specific cellular andhumoral immune responses. Gene Therapy 1998; 5: 1079.

[0138] 49. Brauner R, Nonoyama M, Laks H, et al. Intracoronaryadenovirus-mediated transfer of immunosuppressive cytokine genesprolongs allograft survival. J Thorac Cardiovasc Surg 1997; 114: 923.

[0139] 50. Räisänen-Sokolowski A, Mottrarn P1, Glysing-Jensen T,Satoskar A, Russell M E. Heart transplants in interferon-y, interleukin4, and interleukiii 10 knockout mice. Recipient environment alters graftrejection. J Clin Invest 1997; 100:2449.

[0140] 51. Räisänen-Sokolowski A, Glysing-Jensen T, Russell M E.Leukocyte-suppressing influences of interleukin (L)-10 in cardiacallografts. Insights from IL-10 knockout mice. Am J Pathol 1998; 153:1491.

[0141] 52. Qian S, Li W, Li Y, et al. Systemic administration ofcellular interleukin-10 can exacerbate cardiac allograft rejection inmice. Transplantation 1996; 62: 1709.

[0142] 53. Torres P F, de Vos A F, Martins B, Kijlstra A. Interleukin 10treatment does not prolong experimental corneal allograft survival.Ophthalmic Res 1999 31:2 97. Martin H M, Nash A D, Andrews A E. Cloningand characterisation of an ovine interleukin-10-encoding cDNA. Gene1995; 159: 187.

[0143] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method of modifying cells of corneal tissue toexpress an active agent comprising exposing harvested corneal tissue toan effective concentration for transfection of an expression vectorwhich comprises a nucleotide sequence encoding for the active agent fora period sufficient to allow transfection, such that cells of saidcorneal tissue will express the active agent.
 2. The method according toclaim 1, wherein the nucleotide sequence consists of DNA.
 3. The methodaccording to claim 1, wherein the active agent is selected from thegroup consisting of a peptide hormone, a cytokine and an analoguethereof.
 4. The method according to claim 1, wherein the active agent isselected from the group consisting of an interleukin, an interferon, agrowth factor and an analogue thereof.
 5. The method according to claim3, wherein the cytokine is selected from the group consisting of IL-10,IL-4, the P-40 component of IL-12, Bc12, interferon Gamma, interferonAlpha and TGF Beta.
 6. The method according to claim 1, wherein thecorneal tissue is harvested from a mammal.
 7. The method according toclaim 6, wherein the mammal is a human.
 8. The method according to claim1, wherein the corneal tissue cells modified are selected from the groupconsisting of epithelial cells, stroma cells, endothelial cells andcombinations thereof.
 9. The method according to claim 1, wherein thecorneal cells modified are endothelial cells.
 10. The method accordingto claim 9, wherein at least 5% of a sample of corneal endothelial cellsare modified.
 11. The method according to claim 9, wherein at least 10%of a sample of corneal endothelial cells are modified.
 12. The methodaccording to claim 9, wherein at least 20% of a sample of cornealendothelial cells are modified.
 13. The method according to claim 9,wherein at least 30% of a sample of corneal endothelial cells aremodified.
 14. The method according to claim 9, wherein at least 50% of asample of corneal endothelial cells are modified.
 15. The methodaccording to claim 9, wherein at least 70% of a sample of cornealendothelial cells are modified.
 16. The method according to claim 1,wherein the effective concentration for infection is between about 1×10⁵to 1×10¹⁰ particle forming units (PFU) per cornea.
 17. The methodaccording to claim 1, wherein the effective concentration for infectionis between about 5×10⁵ to 5×10⁸ PFU per cornea.
 18. The method accordingto claim 1, wherein the effective concentration for infection is betweenabout 2×10⁶ and about 9×10⁷ PFU per cornea.
 19. The method according toclaim 1, wherein the period sufficient to allow infection is betweenabout 1 minute and about 48 hours.
 20. The method according to claim 1,wherein the period sufficient to allow infection is between about 10minutes and about 24 hours.
 21. The method according to claim 1, whereinthe period sufficient to allow infection is between about 30 minutes andabout 6 hours.
 22. The method according to claim 1, wherein the periodsufficient to allow infection is between about 1 hour and about 3 hours.23. The method according to claim 1, wherein the expression vector isselected from the group consisting of a viral, a bacterial and a plasmidvector.
 24. The method according to claim 1, wherein the expressionvector is an adeno-associated viral vector or an adenoviral vector. 25.A corneal tissue comprising cells modified to express an active agentwhich is not expressed by normal corneal tissue or which followingmodification is expressed at elevated levels relative to normal cornealtissue.
 26. The corneal tissue according to claim 25, wherein the activeagent is selected from the group consisting of a peptide hormone, acytokine and an analogue thereof.
 27. The corneal tissue according toclaim 25, wherein the active agent is selected from the group consistingof an interleukin, an interferon, a growth factor or an analoguethereof.
 28. The corneal tissue according to claim 25, wherein theactive agent is selected from the group consisting of IL-10, IL-4, theP-40 component of IL-12, PC12, interferon Gamma, interferon Alpha andTGF Beta.
 29. The corneal tissue according to claim 25, wherein thecells modified to express an active agent are selected from the groupconsisting of epithelial cells, stroma cells, endothelial cells andcombinations thereof.
 30. The corneal tissue according to claim 25,wherein the cells modified to express an active agent are endothelialcells.
 31. A corneal tissue comprising cells modified to express anactive agent, wherein modification is by exposing harvested cornealtissue to an effective concentration for infection of an expressionvector which comprises a nucleotide sequence encoding for the activeagent, for a period sufficient to allow infection.
 32. A corneal tissuecomprising cells modified to express an active agent produced accordingto the method of claim
 1. 33. A method of improving corneal grafthealing and/or prolonging graft survival comprising exposing harvestedcorneal tissue to an effective concentration for infection of anexpression vector which comprises a nucleotide sequence encoding for anactive agent for a period sufficient to allow infection, such that cellsof said corneal tissue will express the active agent, and thentransplanting the corneal tissue to an eye of a recipient.
 34. Anexpression vector for use in modifying corneal tissue to express anactive agent not expressed by normal corneal tissue or which followingmodification is expressed at elevated levels relative to normal cornealtissue; the vector comprising a nucleotide sequence encoding for theactive agent.
 35. The expression vector according to claim 34, whereinthe nucleotide sequence is DNA.
 36. The expression vector according toclaim 34, wherein the active agent is selected from the group consistingof a peptide hormone, a cytokine or an analogue thereof.
 37. Theexpression vector according to claim 34, wherein the active agent isselected from the group consisting of an interleukin, an interferon, agrowth factor or an analogue thereof.
 38. The expression vectoraccording to claim 34, wherein the cytokine is selected from IL-10, L-4,the P-40 component of IL-12, Bc12, interferon Gamma, interferon Alphaand TGF Beta.
 39. The expression vector according to claim 34, selectedfrom the group consisting of a viral, a bacterial or a plasmid vector.40. The expression vector according to claim 34, wherein the vector isan adeno-associated viral vector or an adenoviral vector.